The present invention relates to a method for treating cardiac muscle disorders. In particular, the present invention relates to a method for treating cardiac arrhythmia by administration of a neurotoxin to cardiac muscle.
The pumping action of the heart is controlled by sympathetic and parasympathetic (primarily vagal) nerves which abundantly innervate the heart. Heart rate can be increased by sympathetic stimulation and decreased by vagal stimulation. Additionally, many cardiac fibers, such as the sinus node (also called sinoatrial or SA node) have the capability of self-excitation. Stimulation of the sympathetic nerves causes release of norepinephine at the sympathetic nerve endings. Contrarily, stimulation of the parasympathetic nerves to the heart causes acetylcholine to be released at the vagal nerve endings. Hence, the parasympathetic nervous system is often referred to as a cholinergic system.
The release of acetylcholine by the postganglionic parasympathetic nerve endings, by acting upon the muscarinic receptors present in cardiac muscle tissue, as indicated, decreases the rate of rhythm of the sinus node and decreases the excitability of the AV junctional fibers between the atrial musculature and the AV node, thereby slowing transmission of the cardiac impulse into the ventricles. The major site of action of parasympathetic control of the heart appears to be the sinoatrial node, where it reduces the heart rate in contrast to sympathetic stimulation. Other lesser parasympathetic activities include inhibition of the AV node and a mild inhibitory effect on contractile force.
In athletes, parasympathetic activity can increase to slow the heart rate. With excessive physical training, the AV node can be inhibited to block the conduction of the impulse from the SA node to the ventricles, resulting in the condition referred to as AV block.
Notably, all preganglionic neurons are cholinergic in both the sympathetic and parasympathetic nervous systems. Therefore acetylcholine or acetylcholine like substances when applied to the ganglia will excite both sympathetic and parasympathetic postganglionic neurons. Additionally, all or almost all of the postganglionic neurons of the parasympathetic nervous system are also cholinergic. Acetylcholine activates two types of receptors, muscarinic and nicotinic receptors. Parasympathomimetic drugs such as pilocarpine and methacholine mimic the effect of acetylcholine.
Arrhythmia
Cardiac muscle disorders, such as arrhythmias and fibrillations, can result in incapacitation and death. During ventricular fibrillation, the ventricles can quiver in an irregular chaotic way so that little blood is pumped out of the heart and the body, particularly the brain, is deprived of oxygen. During ventricular tachycardia, the heart beats too fast because of rapid electrical impulses starting in the ventricles, which also decreases bloodflow and deprives the body of oxygen.
Natural cardiac rhythms are maintained through the cooperation of sympathetic nerves, which can increase the rate at which the heart beats, and the vagus nerve which can slow down the rate at which the heart beats.
Electrochemical messages from the sympathetic and vagal nerves reach the heart's natural pacemaker, the sinoatrial node, progress to the upper chambers (the atria), and pause at the atrioventricle node, before entering the main pumping chambers, the ventricles. Any breach of this electricalchemical circuit can cause the heart to lurch into a chaotic rhythm.
Arrhythmias are caused by a disruption of the normal functioning of the electrical conduction system of the heart. Normally, the chambers of the heart (atria and ventricles) contract in a coordinated manner. The signal to contract is an electrical impulse that begins in the sinoatrial node (sinus or SA node). This impulse is conducted through the atria and stimulates them to contract. The impulse passes through the atrioventricular node, then travels through the ventricles and stimulates them to contract. Problems can occur anywhere along the conduction system, causing various arrhythmias. Problems can also occur in the heart muscle itself, causing it to respond differently to the signal to contract, also causing arrhythmias, or causing the ventricles to contract independently of the normal conduction system.
Arrhythmias include tachycardias, bradycardias and true arrhythmias of disturbed rhythm. Arrhythmias are classified as lethal if they cause a severe decrease in the pumping function of the heart. When the pumping function is severely decreased for more than a few seconds, blood circulation is essentially stopped, and organ damage (such as brain damage) can occur within a few minutes. Lethal arrhythmias include ventricular fibrillation, also ventricular tachycardia that is rapid and sustained, or pulseless, and may include sustained episodes of other arrhythmias. Additional types of arrhythmias include atrial fibrillation or flutter, multifocal atrial tachycardia, paroxysmal supraventricular tachycardia, Wolff-Parkinson-White syndrome, sinus tachycardia, sinus bradycardia, bradycardia associated with heart block, sick sinus syndrome, and ectopic heartbeat.
In sinus arrhythmia there are cyclic changes in the heart rate during breathing. In sinus tachycardia the sinus node sends out electrical signals faster than usual, speeding up the heart rate. In sick sinus syndrome the sinus node does not fire its signals properly, so that the heart rate slows down. Sometimes the rate changes back and forth between a slow (bradycardia) and fast (tachycardia) rate. With premature supraventricular contractions or premature atrial contractions (PAC) a heart beat occurs early in the atria, causing the heart to beat before the next regular heartbeat. In supraventricular tachycardia (SVT) and paroxysmal atrial tachycardia (PAT) a series of early beats in the atria speed up the heart rate (the number of times a heart beats per minute). In paroxysmal tachycardia repeated periods of very fast heartbeats begin and end suddenly. In atrial flutter there are rapidly fired signals which cause the heart muscles in the atria to contract quickly, leading to a very fast, steady heartbeat. In atrial fibrillation electrical signals in the atria are fired in a very fast and uncontrolled manner. The electrical signals arrive in the ventricles in a completely irregular fashion, so the heart beat is completely irregular. In the Wolff-Parkinson-White syndrome, abnormal pathways between the atria and ventricles cause the electrical signal to arrive at the ventricles too soon and to be transmitted back into the atria. Thus very fast heart rates may develop as the electrical signal ricochets between the atria and ventricles.
Arrhythmias which originate in the ventricles include premature ventricular complexes (PVC) in which an electrical signal from the ventricles causes an early heart beat that generally goes unnoticed. The heart then seems to pause until the next beat of the ventricle occurs in a regular fashion. In ventricular tachycardia the heart beats fast due to electrical signals arising from the ventricles (rather than from theatria). In ventricular fibrillation electrical signals in the ventricles are fired in a very fast and uncontrolled manner, causing the heart to quiver rather than beat and pump blood.
It is known that some arrhythmias are also caused by some drugs. These include antiarrhythmics, Beta blockers, caffeine, cocaine, psychotropics, and sympathomimetics.
Tests that reveal arrhythmias, and which can differentiate between the different types of arrhythmia, include echocardiogram (ECG or EKG), coronary angiography and electrophysiologic study (EPS), the later requiring cardiac catheterization. An ECG records the changing potentials of the electrical field imparted by the heart. Echocardiography refers to a group of tests that utilize ultrasound to examine the heart and record information in the form of reflected sonic waves. Magnetic resonance imaging can also be used as a noninvasive means to determine, at least to some extent, intracardiac pressures and cardiac anatomy. Further details regarding theses diagnostic procedures can be found in Heart Disease A Textbook of Cardiovascular Medicine, edited by Eugene Braunwald, (1997), two volumes, fifth edition, published by W.B. Saunders Company, the entire contents of which is incorporated herein by reference in its entirety.
Therapy for arrhythmia can include systemic administration (by oral or intravenous routes) of an antiarrhythmic drug, surgical removal of the arrhythmic tissue, and/or or implantation of a defibrillator or pacemaker.
Drug Therapy for Arrhythmia
The traditional treatment for the erratic heartbeat of arrhythmia is oral or intravenous administration of an antiarrhythmic drug. A wide variety of antiarrhythmic drugs, such as amiodarone and sotalol, are known, as set forth in Drugs for the Heart by Lionel H. Opie (1997) published by W. B. Saunders Company, the entire contents of which are incorporated herein by reference in its entirety. Antiarrhythmic drugs are generally classified based on their major effects on the heart. Category IA drugs include quinidine, procainamide and disopyramide. Category IB drugs include lidocaine, mexiletine. Category IC drugs include flecainide and propafenone. Category II drugs include beta- blocking drugs. Category III drugs include amiodarone, ibutilide and sotalol. Category IV drugs include calcium-blocking drugs. The category IA, IC and III antiarrhythmic drugs have the major side effects of torsades de points and sudden death. See e.g. Chapter 7. “Antiarrhythmic Drugs” in Drugs for the Heart, supra and Nattel, S., Comparative Mechanisms of Action of Antiarrhythmic Drugs, Am J. Cardiol, 72: 13F–17F (1993), and Wit, A., Electrophysiological Basis for Antiarrhythmic Drug Action, Clin. Physiol. Biochem. 3: 127–134 (1985), both of which later two publications are incorporated herein in their entireties.
Certain arrhythmias, such as atrial fibrillation, can occur post-operatively and various drugs have been administered both pre-operatively and re-initiated immediately after surgery as an intravenous medication to try and treat this condition. Unfortunately, drugs such as sotalol administered intravenously to treat post-operative atrial fibrillation can cause ventricular pro-arrhythmia. Additionally, conditions such as obstructive lung disease and congestive heart failure limit the use of beta blockers antiarrhythmic drugs such as sotalol.
A significant problem with the use of most if not all antiarrhythmic drugs occurs because antiarrhythmic drugs are typically administered intravenously or intraperitoneally resulting in rapid metabolic clearance rates, with concomitant short duration of effective drug level and low drug efficiency. Furthermore, a number of the antiarrhythmic drugs can also have proarrhythmic effects upon the heart.
Thus, current drug therapy for arrhythmia whether by oral or parenteral administration into the systemic circulation has many drawbacks and deficiencies, including undesired and deleterious systemic side effects (lack of selectivity), short duration of action and substantial antigenicity (drug resistance). Additionally, the antiarrhythmic drugs used are expensive, require the person being treated to remember to take them on at least a daily basis, can render the patient groggy and lethargic and are contraindicated for certain patients.
Bradycardia
Significantly, almost one half of all unexpected cardiac arrests which result in sudden death are caused by bradyarrhythmia. Bradyarrhythmia or synonymously bradycardia can be defined as any disturbance of the heart's rhythm which results in a heart rate of under sixty beats per minute. Bradyarrhythmia may occur without obvious underlying cause and without the existence of a previous event such as a myocardial infarction or pulmonary embolism.
Tragically, it is known that sudden death in heart failure resulting from acute myocardial ischemia or infarction, pulmonary embolism, embolic or hemorrhagic stroke, hyperalemia as well as conduction system disease can all be caused by a prior bradycardiac episode.
Drugs that block the effect of acetylcholine, and hence the inhibitory effect of the vagal nerve on the heart, upon the muscarinic type of cholinergic effector organs include atropine and similar drugs such as homatropine and scopolamine. These drugs do not affect the nicotinic receptor action of acetylcholine on the postganglionic neurons or on skeletal muscle. Atropine has a vagolytic effect that is useful for the management of bradyarrhythmias with atrioventricular (AV) block, particularly with inferior infarction, sinus or nodal bradycardia with hypotension, or bradycardia-related ventricular ectopy. Small doses and careful monitoring are essential since the elimination of vagal inhibition may unmask latent sympathetic overactivity, thereby producing tachycardia.
Unfortunately, while symptomatic sinus bradycardia, sick sinus syndrome and sinoatrial disease can be treated with probanthine or by chronic administration of atropine, the results are unsatisfactory in the long run so that implantation of a cardiac pacemaker is the typical therapeutic choice for chronic bradycardia. Additionally, for AV block with syncope or with excessively slow heart rates, atropine or isoproterenol or transthoracic pacing has been used as an emergency measure, but again, only pending pacemaker implantation. Thus, many drawbacks and deficiencies exist with current therapy for bradycardia.
Surgical Removal of Arrhythmic Tissue
Surgery can be carried out to excise the cardiac tissue causing an arrhythmia where the arrhythmia is unresponsive to antiarrhythmic drug therapy. Although closed approaches, such as by biotome catheterization have been used for some of cardiac surgery such as for example, to remove myxomas, including atrial myxomas, closed approach surgical treatment of arrhythmic cardiac tissue is typically treated by either catheter mediated cryoablation or by radiofrequency ablation. Radiofrequency ablation has been used to treat tachycardias such as supraventricular tachycardias and some ventricular tachycardias.
In radiofrequency ablation a catheter enclosing conductive wires with terminal electrodes near the open end or tip of the catheter is inserted into a patient's body through a vein in the thigh, shoulder, or neck. Upon being threaded intravenously to the heart, the catheter tip is positioned inside the heart next to the abnormal heart tissue that is responsible for the tachycardia. Then, a small amount (about 50 watts) of energy is applied to the heart between the tip electrode and a skin patch that is usually placed behind the left shoulder. This energy heats up and thus dries out the heart tissue that is within about 5 millimeters of the tip. After about 30 to 60 seconds of heating, this tissue is no longer alive and can no longer cause tachycardia. Although the actual ablation takes only a minute, the procedure often takes 4 to 10 hours. The reason being that it is time-consuming to identify the exact tissue in the heart that is responsible for a tachycardia and to make sure that all of the relevant tissue has been ablated completely.
Cardiac arrhythmias treatable by radiofrequency ablation include atrioventricular nodal reentrant tachycardia (AVNRT), atrioventricular tachycardia (AVRT) that uses an accessory bypass tract for retrograde conduction, atrial tachycardias that occur in otherwise-normal hearts and also in hearts that have had prior surgery, atrial flutter, and some kinds of ventricular tachycardia that occur in otherwise-normal hearts. The first three rhythms are often grouped together with the term “supraventricular tachycardia”, although this term can also be used to include atrial flutter and atrial fibrillation.
Subsequent to radiofrequency, a cardioverter-defibrillator is often implanted in the patient to prevent recurrence of subsequent arrhythmia by non-ablated cardiac tissues.
Unfortunately, while radiofrequency ablation can treat, it usually does not cure supraventricular tachycardias, including atrial flutter and atrial fibrillation. When a tachycardia is not controlled by antiarrhythmic drugs and cannot be cured by ablation, the symptoms of the arrhythmia (but not the arrhythmia itself can often be controlled by either intentional destruction of the AV node itself or by ablation of the slow AV nodal pathway. In intentional destruction of the AV node, by AV junctional ablation, the upper and lower chambers of the heart are electrically disconnected and the procedure mandates immediate implantation of a permanent pacemaker. AV nodal ablation is used to control otherwise unresponsive atrial fibrillation.
Ablation of the slow AV nodal pathway is the same procedure used to treat AV nodal reentrant tachycardia. For uncontrollable atrial fibrillation and other supraventricular tachycardias, this procedure offers some of the benefit of AV junctional ablation without the need for implantation of a permanent pacemaker. The slow AV nodal pathway procedure takes advantage of the fact that the heart in most patients has two parts to the AV node. The “fast” AV nodal pathway conducts rapidly but takes a long time to recover enough to conduct the next heart beat. The “slow” AV nodal pathway is a backup pathway that conducts slowly but can recover very quickly. At most heart rates, patients use only the fast pathway. When the heart is beating very rapidly (during vigorous exercise, for example), the slow pathway is used because the fast pathway can't recover fast enough between heart beats. When the slow pathway is removed by ablation, the patient almost never can tell the difference at usual heart rates (even during vigorous exercise to, say, a heart rate of 180-200 beats per minute). If a very rapid heart rate (say, to 250 bpm) occurs in the atria, however, the ventricles will go more slowly than they would with an intact slow pathway.
In older patients, who are the ones who usually develop sustained atrial fibrillation, the fast pathway does not conduct as rapidly as in young people, so the maximum heart rate can often be reduced to a range that is tolerable. Two problems with the slow AV nodal procedure for atria fibrillation are, first, when the procedure is continued until the heart rate in atrial fibrillation is reasonable (say, 130 bpm during infusion of isoproterenol, which speeds up the heart rate), about 20% of patients get complete heart block and require immediate implantation of a permanent pacemaker. Second, patients who have undergone the slow AV nodal procedure often don't feel as well as those who go ahead and have AV junctional ablation and pacemaker insertion. The reason seems to be that the heart rate is still erratic because the ventricular rhythm still follows the irregularly atrial fibrillation. By contrast, patients who have AV junctional ablation and pacemakers have regular rhythms because the pacemakers set the heart rate for them.
Unfortunately, the radiation used during the ablation procedure can potentially cause cancer, especially breast cancer in women.
Hyperlipidemia doesn't affect the supraventricular rhythms that are the usual targets of ablation. When ablation is used for the type of ventricular tachycardia that occurs in people who have had myocardial infarction, however, control of hyperlipidemia is quite important to prevent recurrent infarction.
Finally, arrhythmia can also be treated by implantation of a cardiac defibrillator or by implantation of an artificial pacemaker. A cardiac defibrillator is surgically implanted beneath the skin of a patient's abdomen and connected by wires to the ventricles. When arrhythmia occurs, the defibrillator sends an electrical charge to the heart in an attempt to restore normal heartbeat. A defibrillator does not prevent the onset of arrhythmia, but merely attempts to restore the heart's normal rhythm by providing an electric shock to the heart to disrupt an ongoing arrhythmia. Importantly, both defibrillators and pacemakers can malfunction and misfire due, for example to the effect of proximity to an airport metal detector or store security check out device. Furthermore, significant drawback to the use of both defibrillators and pacemakers include the requirement for surgery to implant with attendant risks such as infection.
Angina
A commonly prescribed drug for angina is nitroglycerin, which relieves pain by widening blood vessels. More blood can thereby flow to the heart muscle and the work load of the heart is decreased. Nitroglycerin can be administered when discomfort occurs or is expected. Other drugs to treat angina include beta blockers to slow the heart rate and lessen the force of the heart muscle contraction and calcium channel blockers for reducing the frequency and severity of angina attacks.
Botulinum Toxin
The anaerobic, gram positive bacterium Clostridium botulinum produces a potent neurotoxin, botulinum toxin, which causes a neuroparalytic illness in humans and animals referred to as botulism. The spores of Clostridium botulinum can grow in improperly sterilized and sealed food containers of home based canneries, which are the cause of many of the cases of botulism. The effects of botulism typically appear 18 to 36 hours after eating the foodstuffs infected with a Clostridium botulinum culture. The botulinum toxin passes unattenuated through the lining of the gut and attacks the central nervous system. Symptoms of botulinum intoxication progress from difficulty walking, swallowing, and speaking to paralysis of the respiratory muscles, resulting in suffocation and death.
Botulinum toxin is the most lethal natural biological agent known to man. It has been determined that 39 units per kilogram of intramuscular BOTOX® is a LD50 in primates. One unit (U) of botulinum toxin can be defined as the LD50 upon intraperitoneal injection into mice. BOTOX® contains 4.8 ng of botulinum toxin type A per 100 unit vial. Thus, for a 70 kg human a LD50 of 39 U/kg would be about 131 ng or 27.3 vials (2730 units) of intramuscular BOTOX®. Seven immunologically distinct botulinum toxins have been characterized, being respectively botulinum toxin serotypes A, B, C1, D, E, F and G each of which is distinguished by neutralization with type-specific antibodies. The neurotoxin component is noncovalently bound to nontoxic proteins to form high molecular weight toxin complexes. The different serotypes of botulinum toxin vary in the animal species that they affect and in the severity and duration of the paralysis they evoke. Botulinum toxin type A is the most potent of the seven known serotypes produced by the Clostridium botulinum bacteria and has, in minute quantities, become an important pharmaceutical for the treatment of various segmental and peripheral movement disorders associated with muscle overactivity, such as spasticity, as well as pain, and various other neuronal disorders.
At a normal neuromuscular junction, a nerve impulse triggers the release of acetylcholine, which causes the muscle to contract. Hyperactive muscle contraction is characterized by excessive release of acetylcholine at the neuromuscular junction. The use of a botulinum toxin can be effective in reducing the excessive activity by blocking the release of acetylcholine at the neuromuscular junction.
Botulinum toxin is known to act to reduce excess muscle (both skeletal and smooth muscle) and sphincter contraction and to reduce certain glandular activities upon direct injection into the hyperactive or hypertonic muscle or gland and is believed to exert its effect by entering peripheral nerve terminals at the neuromuscular junction and by blocking the release of acetylcholine. Affected terminals are inhibited from stimulating muscle contraction or inducing glandular activity, resulting in a reduction of muscle tone or reduce secretory output by the targeted gland. Thus, when injected intramuscularly at therapeutic doses, botulinum toxin produces a localized chemical denervation and hence a localized weakening or paralysis and relief from excessive involuntary muscle contractions. When the muscle is chemically denervated, it atrophies and may then in response develop extrajunctional acetylcholine receptors.
Clinical effects of peripheral intramuscular botulinum toxin are usually seen within one week of injection. The typical duration of symptomatic relief from a single intramuscular injection average about three months or longer. Muscles therapeutically treated with a botulinum toxin eventually recover from the temporary paralysis induced by the toxin, due possibly to the development of new nerve sprouts or to reoccurrence of neurotransmission form the original synapse, or both. A nerve sprout establishes a new neuromuscular junction. Thus, neuromuscular transmission can gradually return to normal over a period of several months with no lasting side effects.
Botulinum toxin has no appreciable affinity for organs or tissues other than cholinergic neurons and when it does bind to neuronal receptors, its only known action is to block acetylcholine release without causing neuronal cell death. Botulinum toxin has therefore been used to treat a variety of disorders of cholinergic nervous system transmission.
Botulinum toxins have been used for the treatment of an increasing array of neurologic disorders, most of which are characterized by hyperactive neuromuscular activity in specific focal or segmental muscle regions. Thus intramuscular or intraglandular injection of one or more of the botulinum toxin serotypes has been used to treat, blepharospasm, spasmodic torticollis, hemifacial spasm, spasmodic dysphonia, oral mandibular dystonia and limb dystonias, myofacial pain, headache, bruxism, achalasia, trembling chin, spasticity, juvenile cerebral palsy, hyperhydrosis, excess salivation, non-dystonic tremors, cosmetic treatment of brow furrows, focal dystonias, spasticity, tension headache, migraine headache and lower back pain. Not infrequently, a significant amount of pain relief has also been experienced. These benefits have been observed after local intramuscular injection of, most commonly botulinum toxin type A, or one or another of the other botulinum neurotoxin serotypes.
The following list sets forth total (not per kg of patient weight) units of administrations of BOTOX® that have been used for therapeutic intramuscular injections. The list therefore provides guidelines for the unit amount of BOTOX® that can be used to denervate other, not listed, cholinergic muscles or muscle elements of similar size, such as cardiac muscle tissues and cardiac muscles elements. Thus, it is known that:                (1) about 75–125 units of BOTOX® per intramuscular injection (multiple muscles) can be used to effectively treat cervical dystonia;        (2) 5–10 units of BOTOX® per intramuscular injection can be used to effectively treat glabellar lines (brow furrows) (5 units injected intramuscularly into the procerus muscle and 10 units injected intramuscularly into each corrugator supercilii muscle);        (3) about 30–80 units of BOTOX® can be used to effectively treat constipation by intrasphincter injection of the puborectalis muscle;        (4) about 1–5 units per muscle of intramuscularly injected BOTOX® can be used to effectively treat blepharospasm by injecting the lateral pre-tarsal orbicularis oculi muscle of the upper lid and the lateral pre-tarsal orbicularis oculi of the lower lid.        (5) to treat strabismus, each extraocular muscle to be treated can be injected intramuscularly with between about 1–5 units of BOTOX®, the amount injected varying based upon both the size of the muscle to be injected and the extent of muscle paralysis desired (i.e. amount of diopter correction desired). A maximum dose per intramuscular injection should not exceed 25 U.        (6) to treat upper limb spasticity following stroke by intramuscular injections of BOTOX® into five different upper limb flexor muscles, as follows:                    (a) flexor digitorum profundus: 7.5 U to 30 U            (b) flexor digitorum sublimus: 7.5 U to 30 U            (c) flexor carpi ulnaris: 10 U to 40 U            (d) flexor carpi radialis: 15 U to 60 U            (e) biceps brachii: 50 U to 200 U. Each of the five indicated muscles can be injected at the same treatment session, so that the patient receives from 90 U to 360 U of upper limb flexor muscle BOTOX® by intramuscular injection at each treatment session.                        
Botulinum serotypes B, C1, E and F demonstrate a lower potency than BOTOX® and would therefore be used in greater amounts.
Botulinum toxin type A can be obtained by establishing and growing cultures of Clostridium botulinum in a fermenter and then harvesting and purifying the fermented mixture in accordance with known procedures. Botulinum toxin type A is available from several commercial sources, including Allergan, Inc., of Irvine, Calif. under the tradename BOTOX® Botulinum Toxin Type A purified complex and from Porton Products, Ltd., U.K. under the trade name DYSPORT.
Dickson, in J. Exper Med 37, 711–311 (1923) disclosed that the initial vagal nerves stimulation required to induce the physiological response of fewer heart beats per unit time was about eight times higher in botulinum intoxicated cats than it was in non-botulinum intoxicated cats.
All the botulinum serotypes are initially synthesized as inactive single chain proteins which must be cleaved or nicked by proteases to become neuroactive. The bacterial strains that make serotypes A and G possess endogenous proteases and these serotypes are therefore recovered from bacterial cultures predominantly in their active from. In contrast, types C1, D and E are synthesized by nonproteolytic strains and are therefore unactivated when recovered from culture. Serotypes B and F are produced by both proteolytic and nonproteolytic strains and may be recovered in either the active or inactive form. However, even the proteolytic strains that produce the type B serotype only cleave a portion of the toxin produced. The exact proportion of nicked to unnicked molecules depends on the length of incubation and the temperature of the culture. Therefore, a certain percentage of any preparation of type B toxin is likely to be inactive. The presence of inactive botulinum toxin molecules in a clinical preparation will contribute to the overall protein load of the preparation, which has been linked to increased antigenicity, without contributing to its clinical efficacy.
Although all botulinum toxins serotypes inhibit acetylcholine release at the neuromuscular junction, they do so by affecting different neurosecretory proteins and/or cleaving these proteins at different sites. For example, botulinum types A and E both cleave the 25 kiloDalton (kD) synaptosomal associated protein (SNAP-25), but they target different amino acid sequences within this protein. Botulinum toxin types B, D, F and G act on vesicle-associated protein (VAMP, also called synaptobrevin), with each serotype cleaving the protein at a different site. Finally, botulinum toxin type C1 has been shown to cleave both syntaxin and SNAP-25. These differences in mechanism of action may affect the relative potency and/or duration of action of the various botulinum toxin serotypes.
The size of an active botulinum toxin protein is determined by both the size of the neurotoxin-molecule (150 kD for all serotypes) and its associated non-toxin proteins, which vary widely between serotypes. Type A is produced in both a 900 kD and a 500 kD form. Types B and C1 as a 500 kD complex only. Type D as both a 300 kD and 500 kD form. And types E and F as approximately 300 kD complexes only. Larger complexes contain hemaglutinin and a non-toxic nonhemaglutinin protein that improve the stability of the toxin molecule for oral absorption. It is possible that the larger complexes may have a slower rate of diffusion away from a site of injection.
Acetylcholine
Almost invariably, only a single type of small molecule neurotransmitter is released by each type of neuron in the mammalian nervous system. The neurotransmitter acetylcholine is secreted by neurons in many areas of the brain, but specifically by the large pyramidal cells of the motor cortex, by several different neurons in the basal ganglia, by the motor neurons that innervate the skeletal muscles, by the preganglionic neurons of the autonomic nervous system (both sympathetic and parasympathetic), by the postganglionic neurons of the parasympathetic nervous system, and by some of the postganglionic neurons of the sympathetic nervous system. Essentially, only the postganglionic sympathetic nerve fibers to the sweat glands, the piloerector muscles and a few blood vessels are cholinergic and most of the postganglionic neurons of the sympathetic nervous system secret the neurotransmitter norepinephine. In most instances acetylcholine has an excitatory effect. However, acetylcholine is known to have inhibitory effects at some of the peripheral parasympathetic nerve endings, such as inhibition of the heart by the vagus nerves.
The efferent signals of the autonomic nervous system are transmitted to the body through either the sympathetic nervous system or the parasympathetic nervous system. The heart receives many sympathetic nerve fibers from the neck portion of the sympathetic chain. The preganglionic neurons of the sympathetic nervous system extend from preganglionic sympathetic neuron cell bodies located in the intermediolateral horn of the spinal cord. The preganglionic sympathetic nerve fibers, extending from the cell body, synapse with postganglionic neurons located in either a paravertebral sympathetic ganglion or in a prevertebral ganglion. Since, the preganglionic neurons of both the sympathetic and parasympathetic nervous system are cholinergic, application of acetylcholine to the ganglia will excite both sympathetic and parasympathetic postganglionic neurons.
Specific application of acetylcholine to the preganglionic sympathetic neurons which innervate the heart can result in tachycardia, as well as an increased force of contraction of the heart. Contrarily, specific application of acetylcholine to the preganglionic parasympathetic neurons which innervate the heart can result in bradycardia (as well as a decreased force of contraction of the heart, especially of the atria), the same bradycardiac result being obtained by application of acetylcholine to the postganglionic parasympathetic neurons which reside on or within cardiac muscle.
Acetylcholine activates two types of receptors, muscarinic and nicotinic receptors. The muscarinic receptors are found in all effector cells stimulated by the postganglionic neurons of the parasympathetic nervous system, as well as in those stimulated by the postganglionic cholinergic neurons of the sympathetic nervous system. The nicotinic receptors are found in the synapses between the preganglionic and postganglionic neurons of both the sympathetic and parasympathetic. The nicotinic receptors are also present in many membranes of skeletal muscle fibers at the neuromuscular junction.
What is needed therefore is a method for treating cardiac arrhythmia such as bradycardia and tachycardia without the numerous drawbacks and deficiencies of: (1) antiarrhythmic drug treatments, such as systemic effects, lack of specificity, and short duration of activity; (2) surgical, cryo or radiofrequency ablation, and; (3) which has a longevity of efficacy which can remove or significantly reduce the need for implantation of a pacemaker in order to substantially restore the heart's natural rhythm.